ES2384134T3 - Use of secreted protein products for prevention and treatment of pancreatic diseases and / or obesity and / or metabolic syndrome - Google Patents

Abstract

This invention relates to the use of secreted SF01-SF13 proteins, to the use of polynucleotides encoding these, and to the use of effectors/modulators thereof in the diagnosis, study, prevention, and treatment of pancreatic diseases (e.g. diabetes mellitus), obesity and/or metabolic syndrome and to the use in regeneration of tissues such as pancreatic tissues and others.

Description

Use of secreted protein products for prevention and treatment of pancreatic diseases and / or obesity and / or metabolic syndrome.

This invention relates to the use of secreted SF06 proteins, and to the use of polynucleotides encoding them, in the diagnosis and study of diabetes, obesity and / or metabolic syndrome.

Many human proteins serve as pharmaceutically active compounds. Several classes of human proteins that serve as such active compounds include hormones, cytokines, cell growth factors, and cell differentiation factors. Most of the proteins that can be used as pharmaceutically active compounds fall within the family of secreted proteins. Secreted proteins are generally produced inside the cells of the rough endoplasmic reticulum, then exported to the Golgi complex, and then moved to vesicles or secretory granules, where they are secreted to the outside of the cell by exocytosis. Examples of commercially used secreted proteins are human insulin, thrombolytic agents, interferons, interleukins, colony stimulating factors, human growth hormone, transforming growth beta factor, tissue plasminogen activator, erythropoietin, and various other proteins. Secreted protein receptors, which are membrane-bound proteins, also have potential as therapeutic or diagnostic agents. Therefore, it is important for the development of new pharmaceutical compounds to identify secreted proteins that can be tested for activity in a variety of animal models. Thus, taking into account the dominant role of secreted proteins in human physiology, there is a need to identify and characterize new functions for secreted human proteins and the genes that encode them. This knowledge will allow to detect, treat, and prevent diseases, disorders, and / or medical conditions by the use of secreted proteins or the genes that encode them.

The pancreas is an essential organ that has both an exocrine function involved in the supply of enzymes to the digestive tract and an endocrine function by which various hormones are secreted into the bloodstream. Exocrine function is ensured by acinar and centroacinare cells that produce various digestive enzymes and intercalated ducts that transport these enzymes in alkaline solution to the duodenum. The functional unit of the endocrine pancreas is the islet of Langerhans. The islets are scattered throughout the exocrine portion of the pancreas and are composed of four types of cells: alpha, beta, delta and PP cells, reviewed for example in Kim S.K. and Hebrok M., (2001) Genes Dev. 15: 111-127. Beta cells produce insulin, represent the majority of endocrine cells and form the nucleus of the islets, while alpha cells secrete glucagon and are located in the periphery. Delta cells and PP cells are less numerous and secrete somatostatin and pancreatic polypeptide, respectively.

Initial pancreatic development has been well studied in different species, including chickens, zebrafish, and mice (for a detailed review see Kim & Hebrok, 2001, supra). The pancreas develops from different dorsal and ventral primordia. The development of the pancreas requires the specification of pancreatic primordia along both anterior-posterior and dorsal-ventral axes. Various factors, which are critical for proper pancreatic development have been identified (see Kim & Hebrok, 2001, supra; Wilson M.E. et al., (2003) Mech Dev. 120: 65-80).

In post-natal / adult humans, acinar and ductal cells retain an important proliferative capacity that can ensure cell renewal and growth, while islet cells become mostly mitotically inactive. This is in contrast to rodents, in which the replication of beta cells is an important mechanism in the generation of new beta cells. It has been suggested that, during the development of the embryo, the pancreatic islets of Langerhans originate from the differentiation of ductal cells or other cells with epithelial morphology (Bonner-Weir S. and Sharma A., (2002) J Pathol. 197: 519-526; Gu G. et al., (2003) Mech Dev. 120: 35-43). In adult humans, new beta cells arise in the vicinity of the ducts (Butler A.E. et al., (2003) Diabetes 52: 102-110; Bouwens L. and Pipeleers D.G., (1998) Diabetologia 41: 629-633). However, an intra-islet location or bone marrow origin for adult beta cell precursor cells has also been suggested (Zulewski H. et al., (2001) Diabetes 50: 521-533; lanus A. et al., (2003) J Clin Invest. 111: 843-850). Zulewski H. et al. (2001) Diabetes 50: 521-533; lanus A. et al., (2003) J Clin Invest. 111: 843850). The growth of pancreatic islets is dynamic and responds to changes in the demand for insulin, such as during pregnancy or during the increase in body mass that occurs during childhood. In adults, there is a good correlation between body mass and islet mass (Yoon K.H. et al., (2003) J. Clin. Endocrinol Metab. 88: 2300-2308).

Pancreatic beta cells secrete insulin, which is stimulated by elevated blood glucose levels. Insulin, among other hormones, plays a fundamental role in the regulation of fuel metabolism. Insulin leads to glycogen and triglyceride storage and protein synthesis. The entry of glucose into the muscles and fat cells is stimulated by insulin. In patients suffering from diabetes mellitus, the amount of insulin produced by pancreatic islet cells is too low, resulting in high blood glucose levels (hyperglycemia). In type I diabetes, beta cells are lost due to autoimmune destruction. In type II diabetic patients, liver and muscle cells lose their ability to respond to normal blood insulin levels (insulin resistance). Glucose levels

High blood levels (and also high levels of blood lipids) lead to a deterioration of beta cell function and an increase in beta cell apoptosis. It is interesting to note that the rate of beta cell neogenesis does not appear to change in type II diabetics (Butler et al., 2003 supra), thus causing a reduction in the total mass of beta cells over time. Finally, the application of exogenous insulin becomes necessary in type II diabetics.

The improvement of metabolic parameters such as blood sugar and blood lipid levels (eg by dietary changes, exercise, medication or combinations thereof) before the mass of beta cells has fallen below a critical threshold leads to a Relatively rapid restoration of beta cell function. However, after such treatment the endocrine pancreatic function would remain impaired due to the only slightly increased regeneration rate.

In type I diabetics, the life span of pancreatic islets is dramatically shortened due to autoimmune destruction. Treatments that modulate the immune system have been devised and may be able to stop or sharply reduce the destruction of the islets (Raz I. et al., (2001) Lancet 358: 1749-1753; Chatenoud L. et al., (2003 ) Nat Rey Immunol. 3: 123-132). However, due to the relatively slow regeneration of human beta cells, such treatments could be completely satisfactory only to improve the diabetic condition if combined with an agent that can stimulate the regeneration of beta cells.

Thus, for both type I and type II diabetes (early and late phases) there is a need to discover new agents that stimulate the regeneration of beta cells.

Diabetes is a very disabling disease, since medications do not control blood sugar levels well enough to prevent fluctuation between high and low blood sugar levels. Diabetic patients are at risk of major complications, including diabetic ketoacidosis, end stage renal disease, diabetic retinopathy and amputation. There are also a multitude of related conditions, such as metabolic syndrome, obesity, hypertension, heart disease, peripheral vascular disease, and infections, for which people with diabetes are at substantially increased risk. The treatment of these complications contributes to a considerable extent to the enormous cost that diabetes imposes on healthcare systems worldwide.

Obesity is one of the most prevalent metabolic disorders in the world. It is still a poorly understood human disease that has become an important health problem that is increasingly relevant to Western society. Obesity is defined as a body weight greater than 20% in excess of the ideal body weight, which often results in a significant deterioration of health. Obesity can be measured by the body mass index, an indicator of adiposity and fatness. Additional parameters to define obesity are waist circumferences, skin fold thickness and bioimpedance. It is associated with an increased risk of cardiovascular disease, hypertension, type II diabetes mellitus, hyperlipidemia and an increased mortality rate. Obesity is influenced by genetic, metabolic, biochemical, psychological, and behavioral factors and may be caused by different reasons such as non-insulin dependent diabetes, increased triglycerides, increased carbohydrate-linked energy and low energy consumption (Kopelman , PG, (2000) Nature 404: 635-643).

The concept of 'metabolic syndrome' (syndrome x, insulin resistance syndrome, deadly quartet) was first described in 1966 by Camus and reintroduced in 1988 by Reaven (Camus JP, (1966) Rev Rhum Mal Osteoartic 33: 10 -14; Reaven GM, (1988), Diabetes 37: 1595-1607). Today, metabolic syndrome is commonly defined as agglomeration of cardiovascular risk factors such as hypertension, abdominal obesity, elevated blood triglyceride levels and fasting glucose, as well as low levels of HDL blood cholesterol. Insulin resistance significantly increases the risk of metabolic syndrome development (Reaven G., (2002) Circulation 106: 286-288). Metabolic syndrome often precedes the development of type II diabetes and cardiovascular disease (Lakka H.M. et al., (2002) JAMA 288: 2709-2716). Control of blood lipid levels and blood glucose levels is essential for the treatment of metabolic syndrome (see, for example, Santomauro A.T. et al., (1999) Diabetes, 48: 1836-1841).

The molecular factors that regulate food intake and body weight balance are incompletely understood. Although several candidate genes have been described that are supposed to influence the homeostatic system (s) that regulate body mass / weight, such as leptin or the co-activator of the gamma receptor activated by the peroxisome proliferator, the different molecular mechanisms and / or molecules that influence obesity or body weight / body mass regulations are not known.

There is a need in the prior art for the identification of candidate genes that are specifically expressed in the initial development in certain pancreatic tissues. These genes and the proteins encoded by them can provide tools for the diagnosis and treatment of severe pancreatic disorders and related diseases. For this reason, this specification describes secreted proteins that are specifically expressed in pancreatic tissues in the initial stages of development. The use of these genes and proteins in the diagnosis, prevention and / or treatment of pancreatic dysfunctions, such as diabetes, and others is further described.

related diseases such as obesity and / or metabolic syndrome. These proteins and genes are especially useful in regeneration processes, such as the regeneration of pancreas cells.

This report describes secreted factors referred to as SF01-SF13, which are involved in the development and regeneration of the pancreas, and in the regulation of energy homeostasis. SF01-SF13 correspond to mammalian proteins as described in Table 1.

The invention is identified in claim 1. Additionally, the invention relates to the identification of a (poly) peptide involved in the regulation of homeostasis of energy and / or the metabolism of a mammal and the screening of an agent, which modulates / affects the activity of an SF06 polypeptide. Any information about other SF01-05 and SF07-13 proteins is provided for the purpose of comparison only.

The function of SF01, a molecule related to TNF, was previously unknown. SF01 homologues exist in humans, mice and Danio rerio (zebrafish). Human SF01 is expressed in the brain, hippocampus, and islets of Langerhans. In fish, SF01 is expressed in the brain.

SF02 is conserved from Drosophila to humans. SF02 is a vital protein regulated by development. Drosophila SF02 mutants die from neural system defects. In the mouse, SF02 appears to be enriched in the endoderm and embryo, and appears to be present in the pancreas. In humans, SF02 is present in pancreas, liver, thymus, and spleen.

Glyphicans are a family of heparan sulfate proteoglycans (HSPGs) from the surface of glycosylphosphatidylinositol (GPI) anchored cells. SF03 is a member of the glycan family of heparan sulfate proteoglycans, which is fixed to the cell membrane by a GPI anchor. SF03 is mutated in Simpson-Golabi-Behmel syndrome (SGBS). SGBS is characterized by pre- and post-natal overgrowth and is a recessive condition linked to the X chromosome. SF03 is expressed in the embryonic mesodermal lung, liver and kidney tissues and is thought to interact with various growth factors to regulate tissue and organ growth

The SF04 gene is a shared precursor for alpha-microglobulin and bikunin. Alpha-microglobulin is a lipocalin with immunosuppressive properties, and bikunin is a plasma proteinase inhibitor. The SF04 mRNA is strongly transcribed in the hepatic parenchyma, the pancreas, and the intestinal epithelium. Both encoded proteins are present, accordingly, in hepatocytes, pancreas, kidney and developing intestine. Bikunin works as a tumor suppressor.

SF05 is a neural specific serine protease inhibitor, which is expressed in the fully developing CNS in the mouse. SF05 inhibits the tissue-type plasminogen activator of extracellular protease and plasmin, but not thrombin. Mice deficient in SF05 are / were viable and healthy, except for behavioral defects. The mutant SF05 protein is added and causes familial dementia in humans.

SF06 is secreted by the brain, adrenal cortex and adrenocortical tumors. SF06 is involved in the regulation of the secretion of steroidal hormones and the proliferation of adrenocortical cells as an autocrine and / or paracrine factor.

SF07 is a supposed tumor suppressor gene, which is deactivated in hepatocarcinomas, colorectal cancer and non-small cell lung cancers.

SF08 is a carboxypeptidase that has no known enzymatic activity. SF08 is expressed in developing bones and cartilage.

The SF09 protein has a length of 145 amino acids and contains EF-Hand motifs that fix the calcium ion. According to the expressed assembly of sequence tags (EST), SF09 is expressed in many tissues including the pancreas.

The mouse homologue of the human SF10 is an integrin-binding extracellular matrix protein. SF10 mutations are frequent in patients with Smith-Magenis syndrome (SMS), a clinically recognizable congenital anomaly / multiple mental retardation syndrome.

SF11 is a cysteine proteinase inhibitor for cathepsins B and L, which is well characterized. The expression of SF11 is controlled by TGF-beta and EGF in deciduous cultures and by TGF-beta in astrocyte precursors. A glycosylated form of SF11 is necessary for a cellular proliferation of the FGF-2-sensitive neural stem. The combined contribution of FGF-2 and SF11 to the adult dentate gyrus stimulated neurogenesis. Mice deficient in SF11 exhibited reduced tumor growth.

SF12 is an extracellular matrix and plasma glycoprotein. The expression of SF12 in the adult pancreatic islet is mostly confined to blood vessels.

SF13 is a ubiquitously expressed cytosolic peptidyl-prolyl-cis-trans-isomerase, which is inhibited by the immunosuppressive drug cyclosporine. SF13 is a pro-inflammatory factor for T lymphocytes. SF13 sends signals through

of the CD147 receptor (basigin) and is expressed in acinar cells but not in the islet membranes or MIN-6 cells.

Accordingly, the present specification describes secreted proteins with functions in human metabolism, regeneration, and pancreatic development processes. Specific genes and proteins encoded by them are described as well as effectors / modulators thereof involved in the regulation of pancreatic function and metabolism, especially in diseases of the pancreas such as diabetes mellitus, eg, insulin-dependent diabetes mellitus and / or diabetes. Non-insulin dependent mellitus, and / or metabolic syndrome, obesity, and / or related disorders such as coronary heart disease, eating disorders, cachexia, hypertension, hypercholesterolemia (dyslipidemia), liver fibrosis, and / or gallstones. Additionally, specific genes and proteins encoded by them as well as effectors / modulators thereof involved in the regeneration of pancreatic cells or tissues are described, e.g. cells that have exocrine functions such as acinar cells, central cells and / or ductal cells and / or cells that have endocrine functions, particularly the islet cells of Langerhans such as alpha, beta, delta and / or PP cells, more particularly beta cells

In this specification, a screen was used for secreted factors expressed in the developing mammalian (mouse) pancreas, as described in greater detail in the Examples section (see Example 1). This screen identified SF01-SF13 as secreted factors expressed in the pancreas of the developing mouse. The mammalian SF01-SF13 proteins and polynucleotides encoding these, in particular human SF01-SF13, are involved in the conditions and processes mentioned above.

The proteins homologous to SF01-SF13 and nucleic acid molecules that encode them can be obtained from vertebrate species. For use in the present invention, nucleic acids encoding human SF06 protein are particularly preferred, as characterized by SEQ ID Nos: 30 or 32, wherein said nucleic acid molecule is as defined in claim 4.

The role of mammalian SF01-SF13 in mammalian metabolism was validated by analysis of transcript expression in different tissues and by analysis of the role in adipocyte differentiation (see Examples 3 and 4 for more detail).

Studies of determination of the expression profile (see Examples for more detail) confirm the particular relevance of SF02-SF04 as regulators of energy metabolism in mammals.

Micro networks are analytical instruments routinely used in bioanalysis. A microrred has molecules distributed throughout the surface of a solid support, and stably associated with said surface. The term "microrred" refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds in a substrate. Micro-networks of polypeptides, polynucleotides, and / or antibodies have been developed and find use in a variety of applications, such as gene expression monitoring, drug discovery, gene sequencing, gene mapping, bacterial identification, and combinatorial chemistry. A particular area in which the micro networks are applicable is the analysis of gene expression (see Example 4). Network technology can be used to explore the expression of a single polymorphic gene or the expression profile of a large number of related or non-related genes. When the expression of a single gene is examined, networks are used to detect the expression of a specific gene or its variants. When an expression profile is examined, the networks provide a platform to identify genes that are tissue specific, are affected by a substance that is tested in a toxicology test, are part of a signaling cascade, carry out internal functions, or are specifically related to a predisposition, condition, disease, or particular genetic disorder.

Micro networks can be prepared, used, and analyzed using methods known in the art (see, for example, Brennan ™, (1995) US Patent No. 5,474,796; Schena M. et al., (1996) Proc. Natl. Acad. Sci. USA 93: 10614-10619; Baldeschwieler JD et al., (1995) PCT application WO 95/251116; Shalon TD and Brown PO, (1995) PCT application WO 95/35505; Heller RA et al., (1997) Proc. Natl. Acad. Sci. USA 94: 2150-2155; Heller, MJ and Tu E., (1997) US Patent No. 5,605,662). Various types of microgrids are well known and are described in detail in Schena M., ed. (1999); DNA Microarrays, A Practical Approach, Oxford University Press, London).

The longer oligonucleotides or fragments derived from any of the polynucleotides described herein can be used as elements in a micro network. The microrred can be used in transcription imaging techniques, which monitor the relative levels of expression of large numbers of genes simultaneously as described below. Microrred can also be used to identify genetic variants, mutations, and polymorphisms. This information can be used to determine the function of genes, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor the progression / regression of a disease based on gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of diseases. In particular, this information can be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for said patient. For example, therapeutic agents that are highly effective and can be selected

exhibit the minimum number of side effects for a patient, based on his or her pharmacogenomic profile.

As determined by microgrid analysis, SF02, SF03 and SF13 exhibit differential expression in human primary adipocytes. A strong increasing regulation is observed concerning the expression of SF02 and SF03 during the differentiation of human adipocytes (see Figs. 3 and 5) while a strongly decreasing regulation is observed concerning the expression of SF13 during differentiation of adipocytes. humans (see Fig. 9). The SF02 and SF03 proteins in preadipocytes have the potential to intensify adipocyte differentiation, and SF13 protein in preadipocytes has the potential to intensify adipocyte differentiation at a very early stage. For this reason, proteins SF02, SF03, and SF13 could play an essential role in adipogenesis. The results suggest a role of SF02, SF03, and SF13 in the regulation of human metabolism, for example, as effectors / modulators (for example, enhancers or inhibitors) of adipogenesis. Thus, SF02, SF03, and SF13 are strong candidates for the manufacture of pharmaceutical compositions and medications for the treatment of conditions related to human metabolism, such as diabetes, obesity, and / or metabolic syndrome.

Additionally, whole and sectional mounting in situ hybridizations are shown (see Examples and Figures 1, 7, and 8). The nucleic acid sequence encoding the mouse SF01 protein is expressed in the region of the ventral pancreas. The nucleic acid sequence encoding mouse proteins SF05 and SF06 are expressed in pancreatic tissue (see Figs. 7 and 8).

Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid or probe binding complex, as described in Wahl G.M. et al., (1987; Methods Enzymol, 152: 399-407) and Kimmel A.R. (1987; Methods Enzymol. 152: 507-511), and can be used at a defined severity. Preferably, hybridization under severe conditions means that after washing for 1 hour with 1 x SSC and 0.1% SDS at 50 ° C, preferably at 55 ° C, more preferably at 62 ° C and most preferably at 65 ° C, particularly for one hour at 0.2 x SSC and 0.1% SDS at 50 ° C, preferably at 55 ° C, more preferably at 62 ° C and most preferably at 65 ° C, a positive hybridization signal is observed.

In order to express a biologically active protein, the nucleotide sequences encoding the proteins or functional equivalents can be inserted into appropriate expression vectors, that is, a vector containing the elements necessary for transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding proteins and appropriate transcription and translation control elements. Regulatory elements include, for example, a promoter, an initiation codon, a stop codon, an mRNA stability regulator element, and a polyadenylation signal. The expression of a polynucleotide can be ensured by (i) constitutive promoters such as the cytomegalovirus promoter / enhancer region (CMV), (ii) tissue specific promoters such as the insulin promoter (see, Soria B. et al., ( 2000), Diabetes 49: 157-162), the promoter of the SOX2 gene (see Li M. et al., (1998) Curr. Biol. 8: 971-974). The Msi-1 promoter (see Sakaklbara S. and Okano H., (1997) J. Neuroscience 17: 8300-8312), promoter of the alpha-cardia-myosin heavy chain or the promoter of the human atrial natriuretic factor (Klug MG et al., (1996) J. Clin. Invest 98: 216224; Wu J. et al., (1989) J. Biol. Chem. 264: 6472-8479) or (iii) inducible promoters such as the inducible system by tetracycline. Expression vectors may also contain a selection agent or marker gene that confers resistance to antibiotics such as neomycin, hygromycin, or puromycin resistance genes. These methods include in vitro recombinant DNA techniques, synthesis techniques, and in vivo genetic recombination. Such techniques are described in Sambrook J. et al., (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and Ausubel F.M. et al., (1989) Current Protocols In Molecular Biology, John Wiley & Sons, New York, N.Y.

In a further embodiment of the invention, natural, modified or recombinant nucleic acid sequences encoding the SF06 proteins can be linked to a heterologous sequence to encode a fusion protein.

A variety of expression vectors / host systems known in the art that contain and express sequences encoding fusion proteins or proteins can be used. These include, but are not limited to, micro-organisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus, adenovirus, adeno-associated virus, lentivirus, retrovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (eg, Ti plasmids or PBR322); or animal cell systems.

The presence of polynucleotide sequences in a sample can be detected by hybridization and / or amplification of DNA-DNA or DNA-RNA using probes or portions or fragments of said polynucleotides. Assays based on nucleic acid amplification involve the use of oligonucleotides or oligomers based on the specific sequences for the gene in order to detect transformants containing DNA or RNA encoding the corresponding protein. As used herein, the terms "oligonucleotides" or "oligomers" refer to a nucleic acid sequence of at least about 10 nucleotides and which may contain as many as

about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplifier.

A variety of labels and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detection of polynucleotide sequences include oligo-labeling, nick translation, RNA probe end labeling, PCR amplification using a labeled nucleotide, or enzymatic synthesis. These procedures can be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); And U.S. Biochemical Corp., (Cleveland, Ohio).

The presence of SF01-SF13 in a sample can be determined by immunological methods or activity measurement. A variety of protocols for detection and measurement of protein expression, using polyclonal or monoclonal antibodies specific for the protein or reagents to determine the activity of proteins are known in the art. Examples include the enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell classification (FACS). A two-site immunoassay based on monoclonal antibodies using reactive monoclonal antibodies for two non-interfering epitopes in the protein is preferred, but a competitive binding assay can be employed. These and other trials are described, among other places, in Hampton R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) And Maddox D.E. et al. (1983; J. Exp. Med, 158: 1211-1226).

Suitable reporter molecules or labels that may be used include radionuclides, enzymes, fluorescent agents, chemiluminescent or chromogens as well as substrates, co-factors, inhibitors, magnetic particles, and the like. Nucleic acids encoding SF06 proteins can be used to generate modifications in non-human transgenic animals or site-specific genes in cell lines. Non-human transgenic animals can be produced by homologous recombination, in which the normal locus of the genes encoding the proteins of the invention is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like. Modified cells or animals are useful in the study of the function and regulation of SF06 proteins. For example, a series of small deletions and / or substitutions can be made in the genes encoding the proteins in order to determine the role of particular domains of the protein, functions in pancreatic differentiation, etc.

Specific constructs of interest include antisense molecules, which will block the expression of SF06 proteins, or the expression of dominant negative mutations. A detectable marker, such as lac-Z, for example, can be introduced into the gene locus, where increasing regulation of gene expression will result in an easily detected change in the phenotype.

The expression of genes can also be provided in cells or tissues in which they are not normally expressed or at abnormal moments of development. Additionally, by providing the expression of SF06 proteins in cells in which they do not normally occur, it is possible to induce changes in cellular behavior.

DNA constructs for homologous recombination will comprise at least portions of the genes with the desired genetic modification, and will include regions of homology in the target locus. DNA constructs for random integration do not need to include regions of homology to mediate recombination. Conveniently, markers are induced for positive and / or negative selection. Methods for generating cells that have gene-directed modifications by homologous recombination are known in the art. For non-human embryonic stem (ES) cells, an ES cell line can be employed, or embryonic cells can be obtained recently from a host, e.g., mouse, rat, guinea pig, etc. Such cells are grown in an appropriate layer of fibroblast-feeders or cultured in the presence of the leukemia inhibitor factor (LIF).

The data described herein demonstrate that SF06 nucleic acids and proteins are useful in diagnostic applications concerning diabetes (such as insulin-dependent diabetes mellitus and / or non-insulin-dependent diabetes mellitus), obesity, and / or metabolic syndrome. Therefore, the diagnostic uses for nucleic acids and proteins are, for example but not limited to, the following: (i) diagnosis of antibody targets, (ii) diagnostic marker and / or prognosis, and / or (iii) research tools

SF06 nucleic acids and proteins are useful in diagnostic applications involved in various embodiments as described below.

Nucleic acids may be additionally useful in diagnostic applications, where the presence or quantity of nucleic acids or proteins, selected from diabetes, obesity and metabolic syndrome, should be evaluated.

A variety of protocols that include ELISA, RIA, and FACS for protein determination are known in the art and provide a basis for the diagnosis of abnormal or abnormal levels of gene expression. Normal or standard values for gene expression are established by combination of body fluids or extrac

cough of cells taken from normal, preferably human, mammalian individuals with antibodies to the protein under conditions suitable for complex formation. The amount of standard complex formation can be quantified by various methods, but preferably by photometric means. The amounts of protein expressed in the control and disease samples, e.g. of tissues undergoing biopsy are compared with standard values. The deviation between the standard values and those corresponding to individuals establishes the parameters for diagnosis of the disease.

In another embodiment of the invention, specific polynucleotides for SF06 proteins can be used for diagnostic purposes. The polynucleotides that can be used include oligonucleotide sequences, RNA molecules and antisense DNA, and PNAs. Polynucleotides can be used to detect and quantify gene expression in tissues undergoing biopsy in which gene expression can be correlated with the disease. The diagnostic assay can be used to distinguish between absence, presence, and excess gene expression, and to monitor the regulation of protein levels during a therapeutic intervention.

In one aspect, hybridization with probes that are capable of detecting polynucleotide sequences, including genomic sequences, encoding SF06 proteins, can be used to identify nucleic acid sequences encoding the respective protein. The hybridization probes may be DNA or RNA and are preferably derived from the nucleotide sequence of the polynucleotide encoding the SF06 proteins or from a genomic sequence that includes promoter, enhancer elements, and introns of the naturally occurring gene. Hybridization probes may be labeled by a density of reporter groups, for example, radionuclides such as 32P or 35S or enzymatic markers, such as alkaline phosphatase coupled to the probe by avidin / biotin coupling systems, and the like.

Specific polynucleotide sequences for SF06 proteins can be used to diagnose conditions or diseases, which are associated with protein expression, selected from diabetes, obesity and metabolic syndrome. Specific polynucleotide sequences for SF06 proteins can also be used to monitor the progress of patients receiving treatment for diabetes, obesity, and / or metabolic syndrome. The polynucleotide sequences can be used in qualitative or quantitative assays, e.g. in Southern or Northern analysis, spot transfer or other membrane-based technologies; in PCR technologies; or in tests with dip rods, spindles, ELISAs or chips that use fluids or tissues from patient biopsies to detect altered gene expression.

In a particular aspect, SF06 nucleotide sequences may be useful in assays that detect the activation or induction of the above metabolic diseases or dysfunctions. Nucleotide sequences can be labeled by standard methods, and added to a patient's fluid or tissue sample under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared to a standard value. The presence of altered levels of nucleotide sequences encoding the SF06 proteins in the sample indicates the presence of an associated disease. Such trials can also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of a disease associated with the expression of SF06 proteins, a normal or standard profile for expression is established. This can be done by combining body fluids or cell extracts taken from normal individuals, whether animal or human, with a sequence or fragment thereof, which is specific for the nucleic acids encoding SF06 proteins, under appropriate conditions. for hybridization or amplification. Standard hybridization can be quantified by comparison of the values obtained from normal individuals with those from an experiment in which a known amount of a substantially purified polynucleotide is used. The standard values obtained from normal samples can be compared with the values obtained from samples of patients presenting symptoms of disease. The deviation between the standard values and those of the individual in question is used to establish the presence of disease. Once the disease is established and a treatment protocol has been initiated, hybridization assays can be repeated on a regular basis to assess whether the level of expression in the patient begins to approximate that observed in the normal patient. The results obtained from successive trials can be used to demonstrate the effectiveness of the treatment over a period of several days to months.

With respect to diabetes, obesity, and / or metabolic syndrome, the presence of an unusual amount of transcript in the tissue from an individual's biopsy may indicate a predisposition for the development of the disease, or it may provide a means to detect the disease. before the appearance of real clinical symptoms. A more definitive diagnosis of this type may allow health professionals to use preventive measures or aggressive treatment sooner, thereby preventing the development or subsequent progression of metabolic diseases and disorders.

Additional diagnostic uses for oligonucleotides designed from the sequences encoding the SF06 proteins may involve the use of PCR. Such oligomers can be chemically synthesized, generated by enzymatic means or produced from a recombinant source. The oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5prima.fwdarw.3prima) and one with anti orientation

sense (3prima, rarw.5prima), used under conditions optimized for identification of a specific gene or condition. The same two oligomers, nested oligomer sets or even a degenerate cluster of oligomers can be used under less severe conditions for detection and / or quantification of closely related sequences of DNA or RNA.

Nucleic acid sequences can also be used to generate hybridization probes, which are useful for mapping the naturally existing genomic sequence. The sequences can be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. Such techniques include FISH, FACS or artificial chromosome constructs, such as artificial yeast chromosomes, bacterial artificial chromosomes, P1 bacterial constructs or single chromosome cDNA libraries as reviewed in Price CM, (1993) Blood Rev. 7: 127 -134, and Trask BJ, (1991) Trends Genet. 7: 149-154. FISH (as described in Verma R.S. and Babu A., (1989) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York, N.Y.). The results can be correlated with other physical chromosome mapping techniques and genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265: 1981f). The correlation between the location of the gene that encodes the SF06 proteins on a physical chromosomal map and a specific disease or predisposition for a specific disease can help to delimit the region of DNA associated with said genetic disease.

Nucleotide sequences can be used to detect differences in gene sequences between normal, carrier or affected individuals. A polymorphism analysis can be performed, e.g. simple nucleotide polymorphisms. Additionally, in situ hybridization of chromosomal preparations and physical mapping techniques such as link analysis using established chromosomal markers for the extension of genetic maps can be used. Often, the location of a gene on the chromosome of another mammalian species, such as a mouse, may reveal associated markers even if the number or branch of a particular human chromosome is unknown. New sequences can be assigned to chromosomal branches or part thereof, by physical mapping. This provides valuable information for researchers looking for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been located approximately by genetic links to a particular genomic region, for example AT at 11q22-23 (Gatti RA et al., (1988) Nature 336: 577-580), any sequences that map This area may represent associated or regulatory genes for further investigation. Nucleotide sequences can also be used to detect differences in chromosomal location due to translocation, inversion, etc. between normal individuals, carriers or affected.

In another embodiment of the invention, SF06 proteins can be used for screening libraries of compounds in any of a variety of drug screening techniques. It is possible to identify effectors, e.g. receptors, enzymes, proteins, ligands, or substrates that bind to, modulate or mimic the action of one or more of the SF06 proteins. The protein or fragment thereof used in said screening may be free in solution, fixed to a solid support, supported on a cell surface, or located intracellularly. The formation of binding complexes between the SF06 proteins and the tested agent can be measured. Agents could also directly or indirectly influence the activity of SF06 proteins.

Additionally, the activity of SF06 proteins against their or their physiological substrates or derivatives thereof could be measured in cell-based or cell-free assays. Agents may also interfere with post-translational modifications of the protein, such as phosphorylation and dephosphorylation, farnesylation, palmitoylation, acetylation, alkylation, ubiquitination, proteolytic processing, subcellular localization and degradation. In addition, the agents could influence the dimerization or oligomerization of the SF06 proteins or, in a heterologous manner, of the SF06 proteins with other proteins, for example, but not exclusively, coupling proteins, enzymes, receptors, or translation factors . The agents could also act on the physical interaction of SF06 proteins with other proteins, which are required for protein function, for example, but not exclusively, their downstream signaling.

Methods for determining protein-protein interaction are well known in the art. For example, the fixation of a fluorescently labeled peptide derived from the interaction protein to the SF06 protein, or vice versa, could be detected by a change in polarization. In the event that both members of the binding partner, which can be full-length proteins as well as one member of the binding partner such as full-length protein and the other represented just as a peptide are fluorescently labeled, the binding could be detected by fluorescence energy transfer (FRET) from one fluorophore to the other. Additionally, a variety of commercially available assay principles, suitable for detection of protein-protein interaction are well known in the art, for example, but not exclusively, the Alpha Screen (Perkin Elmer) or Proximity Scintillation (SPA) Assays. by Amersham. Alternatively, the interaction of SF06 proteins with cellular proteins could be the basis for a cell-based screening assay, in which both proteins are fluorescently labeled and the interaction of both proteins is detected by cotranslocation analysis of both proteins with a cell imaging reader, as developed, for example, but not exclusively, by Cellomics or EvotecOAI. In all cases, the two or more members of the binding partner can be different proteins, the SF06 protein being one, or in the case of dimerization and / or oligomerization the SF06 protein itself.

Of particular interest are screening assays for agents that have low toxicity to mammalian cells. The term "agent", as used herein, describes any molecule, e.g. protein or pharmaceutical compound, with the ability to alter or mimic the physiological function of one or more of the SF06 proteins. Candidate agents encompass numerous classes of chemicals, although typically they are organic molecules, preferably small organic compounds having a molecular weight greater than 50 and less than about 2500 Daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonds, and typically include at least one amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. Candidate agents often comprise carbocyclic or heterocyclic structures and / or aromatic or polyaromatic structures constituted with one or more of the above functional groups.

Candidate agents are also among biomolecules that include peptides, saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acids and derivatives, structural analogues or combinations thereof. Candidate agents are obtained from a wide variety of sources that include libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds are available in the form of bacterial, fungal, vegetable and animal extracts, or are easily produced. Additionally, libraries and natural or synthetic-produced compounds are easily modified by conventional chemical, physical and biochemical means, and can be used to produce combinatorial libraries. Known pharmacological agents may undergo directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogues. In cases where the screening assay is a binding assay, one or more of the molecules can be attached to a label, where the label can directly or indirectly provide a detectable signal.

Another technique that can be used for drug screening provides high capacity screening of compounds that have adequate binding affinity for the protein of interest, as described in published PCT application WO 84/03564. In this method, as applied to SF06 proteins, large numbers of different small test compounds, eg, aptamers, peptides, low molecular weight compounds etc., are provided or synthesized on a solid substrate, such as plastic spindles or Some other surface. Test compounds are reacted with the proteins or fragments thereof, and washed. The fixed proteins are then detected by methods well known in the art. The purified proteins can also be applied directly as a plaque coating for use in the drug screening techniques mentioned above. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support. Competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound for protein binding can be used. In this way, antibodies can be used to detect the presence of any peptide, which shares one or more antigenic determinants with the protein. Compounds that fix SF06 proteins, e.g. antibodies, are useful for the identification or enrichment of cells, which are positive for the expression of SF06 proteins, from complex mixtures of cells. Such cell populations are useful in transplantation, for experimental evaluation, and as a source of specific lineage and cell products, including mRNA species useful in the identification of genes specifically expressed in these cells, and as a target for the identification of factors. of molecules that can affect them. Cells that express SF06 protein or that have been treated with SF06 protein are useful in transplantation to provide a receptor with pancreatic islet cells, including insulin-producing beta cells; for drug screening; experimental models of differentiation and interaction of islets with other cell types; in vitro screening assays that define growth and differentiation factors, and to further characterize genes involved in the development and regulation of islets; and analogues. For these purposes, native cells can be used, or they can be genetically modified to provide altered capabilities. Cells from a regenerating pancreas, the anterior intestine, embryonic stomach and duodenum, or other sources of progenitor pancreatic cells can be used as the starting population. The progenitor cells can be obtained from any mammalian species, e.g. horses, cattle, pigs, dogs, cats, rodents, e.g. mice, rats, hamster, primates, etc., in particular of the human species.

In another embodiment, in a high potency screening method, the cells are transfected with a DNA construct,

e.g. a viral or non-viral vector containing an reporter gene, e.g. the lacZ gene or the GFP gene, under regulatory control of a promoter of a gene involved in, for example, the differentiation of beta cells, e.g. a promoter of a gene that stimulates differentiation of beta cells, preferably a Pax4 promoter. Transfected cells are divided into aliquots and each aliquot is contacted with a test substance, eg, candidate 1, candidate 2 and candidate 3. The activity of the reporter gene corresponds to the ability of the test compound to induce differentiation. of beta cells.

In a further embodiment, which can be combined with the high power screening described above, a power validation of the medium is performed. In it, the test compound is added to the stem cells that are cultured and insulin production is determined. After an initial high potency assay, such as the cell-based assay outlined above in which for example a Pax4 promoter is used as a marker for the

regeneration of the beta cells, the activity of the candidate molecules is tested to induce the differentiation of the beta cells in a validation test comprising adding said compounds to the culture medium of the embryoid bodies. Differentiation in insulin producing cells is then evaluated, e.g. by comparison with wild-type cells and / or ES cells expressing Pax4 to evaluate the efficacy of a compound.

Nucleic acids encoding SF06 proteins can be used to generate transgenic non-human cell and animal lines. These non-human transgenic animals are useful in the study of the function and regulation of SF06 proteins in vivo. Non-human transgenic animals, particularly transgenic mammalian animals, can serve as a model system for the investigation of many developmental processes and cellular processes common to humans. A variety of nonhuman models of metabolic disorders can be used to test modulators of the SF06 protein. The incorrect expression (for example, overexpression or lack of expression) of the SF06 protein, particularly the feeding conditions, and / or the administration of biologically active compounds can create models of metabolic disorders.

In one embodiment of the invention, such assays use mouse models of insulin resistance and / or diabetes, such as mice bearing gene silencing in the leptin pathway (eg, ob (leptin) or db (receptor) mice. of leptin)), as described above. In addition to testing the expression of SF06 proteins in such mouse varieties (see Examples), these mice could be used to test whether the administration of a candidate modulator alters, for example, the accumulation of lipids in the liver, plasma or tissues. adipose using standard assays well known in the art, such as FPLC, colorimetric assays, blood glucose level tests, insulin tolerance tests, and others.

Non-human transgenic animals can be produced by homologous recombination in embryonic stem cells, where the normal locus of the gene encoding the SF06 protein is mutated. Alternatively, a nucleic acid construct that encodes the protein is injected into oocytes and randomly integrated into the genome. It is also possible to express genes in tissues in which they are not expressed normally or at abnormal moments of development. Additionally, gene variants as specific constructs that express antisense molecules or expression of dominant negative mutations, which will block or alter the expression of SF06 proteins can be randomly integrated into the genome. A detectable marker, such as lacZ or luciferase can be introduced into the gene locus, where increasing regulation of gene expression will result in an easily detectable change in the phenotype. Vectors for stable integration include plasmids, retroviruses and other animal viruses, artificial yeast chromosomes (YACs) and the like. DNA constructs for homologous recombination will contain at least portions of the genes with the desired genetic modification, and will include regions of homology for the target locus. Conveniently, markers for positive and negative selection are included. DNA constructs for random integration do not need to contain regions of homology to mediate recombination. DNA constructs for random integration will consist of nucleic acids that encode proteins, a regulatory element (promoter), an intron and a poly-adenylation signal. Methods for generating cells that have gene modifications directed by homologous recombination are known in the field. For embryonic stem cells (ES), an ES cell line can be used, or embryonic cells can be obtained recently from a host, e.g., mouse, rat, guinea pig, etc. Said cells are grown on an appropriate fibroblast-feeder layer and grown in the presence of leukemia inhibitor factor (LIF). ES or embryonic cells can then be transfected and then used to produce transgenic animals. After transfection, ES cells are plated on a feeder layer in an appropriate medium. Cells containing the construct can be selected using a selection medium. After sufficient time for the colonies to grow, they are selected and analyzed for the existence of homologous recombination. Colonies that are positive can then be used for embryo manipulation and morula aggregation. In short, morbidi of superovulated females 4 to 6 weeks of age are obtained, the Pellucid Zone is separated and the morulas are placed in small depressions of a tissue culture capsule. ES cells are trypsinized, and modified cells are placed in depression very close to the morula. The next day, the aggregates are transferred to the uterine tubes of pseudopregnated females. The females are then allowed to arrive at term. The chimeric offspring can be easily detected by a change in the color of the layer and are subsequently screened for transmission of the mutation to the next generation (F1 generation). The offspring of the F1 generation is screened for the presence of the modified gene and the males and females that present the modification mate to produce homozygous progeny. If the gene alterations cause lethality at some point in development, the tissues or organs can be maintained as grafts or halogenic or congenital transplants, or as an in vitro culture. Transgenic animals can be any non-human mammal, such as a laboratory animal, pets, etc., for example, mouse, rat, guinea pig, sheep, cow, pig, and others. Transgenic animals can be used in functional studies, drug screening, and other applications and are useful in studying the function and regulation of SF06 proteins in vivo.

The figures show:

Fig. 1 shows the results of in situ hybridization for the FS01 protein.

Fig. 1A shows an in situ hybridization of whole assembly of mouse embryonic pancreas on day E11.5 (side view).

Fig. 1B shows a complete assembly of in situ hybridization of mouse embryonic pancreas on day E11.5 (ventral view; after extirpation of the liver; major increase of the stained region).

Fig. 2 shows the expression of SF02 in mammalian tissues (mouse).

Fig. 2A shows the real-time PCR analysis of SF02 expression in wild-type mouse tissues (referred to as wt mice) and with control diet (referred to as controldiet) tissues.

Fig. 2B shows the real-time PCR analysis of SF02 expression in genetically obese mice (referred to as ob / ob mice) compared to wild-type mice and in mice fed a high-fat diet (a those referred to as HFD mice) compared to mice fed the control diet.

Fig. 3 shows the microgrid analysis of SF02 expression in human abdominal adipocyte cells, during preadipocyte differentiation into mature adipocytes.

Fig. 4 shows the expression of SF03 in mammalian tissues (mouse).

Fig. 4A shows the real-time PCR analysis of SF03 expression in wild-type mouse tissues (referred to as wt mice) and with control diet (referred to as controldiet).

Fig. 4B shows the real-time PCR analysis of SF03 expression in genetically obese mice (referred to as ob / ob mice) compared to wild-type mice and mice fed a high-fat diet (at which is referred to as HFD mice) compared to mice fed a control diet.

Fig. 5 shows the microgrid analysis of SF03 expression in human abdominal adipocyte cells during differentiation of preadipocytes to mature adipocytes.

Fig. 6 shows the expression of SF04 in mammalian tissues (mouse).

Fig. 6A shows the real-time PCR analysis of SF04 expression in tissues of wild-type mice (referred to as wt mice) and with control diet (referred to as controldiet) (with liver inclusion).

Fig. 6B shows the real-time PCR analysis of SF04 expression in wild-type mouse tissues and tissues with control diet (without liver).

Fig. 6C shows the real-time PCR analysis of SF04 expression in genetically obese mice (referred to as ob / ob mice) compared to wild-type mice and mice fed a high-fat diet (a those referred to as HFD mice) compared to mice fed a control diet.

Fig. 7 shows the results of in situ hybridization for the SF05 protein.

Fig. 7A shows a cryosection of mouse embryonic pancreas on day E17.5.

Fig. 7B shows the cryosection of mouse embryonic pancreas on day E17.5 with a larger increase.

Fig. 8 shows the results of in situ hybridization for the SF06 protein. Cryosection of mouse embryonic pancreas on day E17.5 is shown.

Fig. 9 shows the microgrid analysis of SF13 expression in human adipocytes during differentiation of preadipocytes to mature adipocytes.

Fig. 9A shows the microgrid analysis of SF13 expression in human primary abdominal adipocytes during preadipocyte differentiation into mature adipocytes.

Fig. 9B shows the microgrid analysis of SF13 expression in human SGBS cells during preadipocyte differentiation into mature adipocytes.

EXAMPLES

Example 1: Identification of secreted factors expressed in pancreas

Screening for secreted factors expressed in developing mouse pancreas was carried out according to methods known to those skilled in the art (see, for example, Pera EM and De Robertis EM, (2000) Mech Dev 96 (2): 183-195) with several modifications.

CDNA expression library:

During organogenesis, the pancreatic primordium is surrounded and influenced by the associated mesenchyme (see, for example, Madsen OD et al., (1996) Eur. J. Biochem. 242: 435-445 and Slack, JM, (1995) Development 121: 1569-1580). Recently, it has been suggested that white adipocytes originate directly from mesenchymal cells (Atanossova P.K., (2003) Folia Med. 45: 41-45). During embryogenesis, innervation and vascularization of the pancreas can be observed. Therefore, the tissue used in screening could have contained in addition to pancreatic cells some precursors of adipocytes, blood vessels, as well as neuronal cells.

A library of mouse pancreatic primordia was prepared in the embryonic phase 9.5-15 in a pCMVSPORT-6 vector using the Invitrogen SUPERSCRIPT Plasmid System according to the manufacturer's instructions. The non-amplified library was electroporated in MaxEff DH10B cells (Invitrogen).

Cloning of the secretion

Bacterial clones with sterile toothpicks were selected from agar plates and cultured in 96-well deep microtiter plates in LB ampicillin (see Sambrook et al., Supra). Aliquots of 8 cultures were pooled, and plasmid DNA was isolated using the BioRobot_9600 apparatus according to the manufacturer's instructions (Qiagen; BioRobot Turbo Kit QIAprep (r)). Human 293 culture cells were cultured in 75 ml tissue culture flasks in DMEM and 10% fetal calf serum. When 90-99% of the confluence was reached, the cells were divided in a 1: 3 ratio and spread on 96-well plates coated with poly-D-lysine (Sigma). Cells were transfected with 100-500 ng of plasmid using lipofectamine 2000 (Invitrogen). After 6 hours, the medium was changed by full means of recent growth. 24 hours after transfection, the cells were washed twice with DMEM without cysteine and methionine (Invitrogen), supplemented with 1% dialyzed bovine serum (Sigma) with 50 micrograms per ml of heparin (Sigma) and glutamine. Cells were radiolabelled ('S35 Met-Label', from Hartman Analytic GmbH). After 12 hours, aliquots of the supernatants were harvested in 96-well PCR plates and subjected to SDS gel electrophoresis in Criterion gels pre-melted in 4020% polyacrylamide gradient (Biorad) under reducing conditions, using an execution chamber in Criterion Dodeca Cell gel (Biorad). The gels were fixed in 10% acetic acid, 25% isopropanol for 30 min, impregnated for 15-30 min in AMPLIFY reagent (Amersham), dried and exposed to X-OMAT (AR) film (Kodak). Positive clones were identified and allowed to grow again in 96-well plates. The DNA of individual clones was prepared and used for transfection as described above. If one of the clones produced proteins of the same size as the original cluster, a positive clone was identified. Positive clones were partially sequenced from the 5 'end (SEQLAB, Goettingen).

Example 2: Identification of homologous human nucleic acid and protein sequences

The term "polynucleotide comprising the nucleotide sequence shown in the GenBank Access number" refers to the expressible gene of the nucleotide sequences deposited under the corresponding GenBank Access number. The expression "GenBank Access number" refers to entries in the NCBI GenBank database (ref .: Benson D.A. et al., (2000) Nucleic Acids Res. 28: 15-18).

Homologous sequences to mouse sequences were identified using the publicly available BLASTP 2.2.3 program from the National Center's non-redundant protein database by Biotechnology Information (NCBI) (see, Altschul SF et al., (1997) Nucleic Acids Res. 25: 3389-3402).

SF01-SF13 proteins and nucleic acid molecules encoding them can be obtained from insect or vertebrate species, e.g. Mammals or fish Particularly preferred are nucleic acid molecules and proteins encoded by them comprising human SF01-SF13 and SF01-SF13 mouse sequences identified in the "secreted factor screening", as described in Table 2.

13 Table 2: Mammalian genes and proteins of the invention (SF01-SF13)

Name

Genbank Access Numbers

Mus musculus genes and proteins

Genes and proteins of Homo sapiens

cDNA

 Protein cDNA Protein 5

Example 3: Analysis of the expression of the nucleic acids described in mammalian tissues (mouse)

To analyze the expression of the mRNAs described herein in mammalian tissues, several mouse varieties (preferably mouse varieties C57BI / 6J, C57BI / 6 ob / ob, C57BI / KS db / db, and mice No- Obese-Diabetics (NOD), which are standard model systems in obesity and diabetes research) were acquired from Harlan Winkelmann (33178 Borchen, Germany) and Taconic M&B (Germantown, NY 12526, USA), 25 respectively, and were they maintained at constant temperature (preferably 22 ° C), 40% humidity and a light / dark cycle of, preferably, 14/10 hours. The mice were fed a standard meal (for example, from Ssniff Spezialitäten GmbH, order number Ssniff M-Z V1126-000). In a subsequent experiment, wild-type (wt) mice were fed a control diet (preferably Altromin C1057 mod control, 4.5% crude fat) or fat-rich diet (preferably Altromin C1057 fat-rich mod, 23 , 5% crude fat). Animals

30 were sacrificed at the age of 6 to 8 weeks. Animal tissues were isolated according to standard procedures known to those skilled in the art, abruptly frozen in liquid nitrogen and stored at -80 ° C until necessary.

To analyze the role of the proteins described in the in vitro differentiation of mammalian cell culture cells for the conversion of preadipocytes into adipocytes, mammalian fibroblast cells (3T3-L1) 35 (eg, Green H. and Kehinde O) were obtained. ., (1974) Cell 1: 113-116) of the American Tissue Culture Collection (ATCC), Hanassas, VA, USA; ATCC-CL 173). 3T3-L1 cells were maintained as fibroblasts and differentiated into adipocytes as described in the prior art (e.g. Qlu Z. et al., (2001) J. Biol. Chem. 276: 11988-11995; Slleker

L.J. et al., (1998) BBRC 251: 225-229). In summary, the cells were extended in DMEM / 10% FCS (Invitrogen, Karlsruhe, Germany) to 50,000 cells / well in duplicate in 6-well plastic capsules and grown in a humidified atmosphere of 5% CO2 at 37 ° C. At the confluence (defined as day 0: d0) the cells were transferred to serum-free medium (SF), which contained DMEM / Ham F12 (3: 1; Invitrogen), fetuine (300 µg / ml; Sigma, Munich, Germany), transferrin (2 µg / ml; Sigma), pantothenate (17 µM; Sigma), biotin (1 µM; Sigma), and EGF (0.8 nM; Hoffmann-La Roche, Basel, Switzerland). Differentiation was induced by the addition of dexamethasone (DEX; 1 µM; Sigma), 3-methyl-isobutyl-1-methylxanthine (MIX; 0.5 mM, Sigma), and bovine insulin (5 µg / ml; Invitrogen ). Four days later

45 of the confluence (d4), the cells were kept in SF medium, containing bovine insulin (5 µg / ml) until differentiation was completed. At various times of the differentiation procedure, beginning on day 0 (confluence day) and day 2 (hormone addition; for example, dexamethasone and 3-isobutyl-1-methylxanthine), up to 10 days of differentiation, were taken aliquots of the cells every 2 days.

RNA was isolated from mouse tissues or cell culture cells using the Trizol Reagent (for example, from Invitrogen, Karlsruhe, Germany) and subsequently purified with the RNAeasy Kit (for example, from Qiagen, Germany) in combination with a DNase treatment according to the manufacturers instructions and as is known to those skilled in the art. Total RNA was subjected to reverse transcription (preferably using RNase H-SuperScript II Reverse Transcriptase, from Invitrogen, Karlsruhe, Germany) and subjected to Taqman analysis, preferably using Mixtura Master Taqman 2 x PCR (from Applied Biosystems, Weiterstadt, Germany ; the Mixture contains, in accordance with the manufacturer, for example Gold AmpliTaq DNA polymerase, AmpErase UNG, dNTPs with dUTP, the Rox passive reference and optimized buffer components) in a GeneAmp 5700 Sequence Detection System (from Applied Biosystems, Weiterstadt, Germany).

The following initiator / probe pairs were used for the Taqman analysis (GenBank Accession Number NM_178644 (mouse) for the mouse SF02 sequence):

Direct mouse SF02 primer (Seq ID NO: 1): 5'- CGG ACA GCA TCA GCC TTG A -3 '; mouse SF02 reverse primer (Seq ID NO: 2): 5'- CCG CGA TGA AGG AGA TGA GA -3 '; Taqman SF02 mouse probe (Seq ID NO: 3): (5/6-FAM) - CTG CGC AAA CCC GAC GGC A - (5/6-TAMRA).

The following initiator / probe pairs were used for the Taqman analysis (GenBank Accession Number NM_016697 (mouse) for the mouse SF03 sequence):

Direct mouse SF03 primer (Seq ID NO: 4): 5'- GTT GTT CGC CAT GCC AAG A -3 '; reverse mouse primer SF03 (Seq ID NO: 5): 5'- CAA AAG CTT GTG GAG TCA GGC T -3 '; Taqman SF03 mouse probe (Seq ID NO: 6): (5/6-FAM) - ACA CCA ACG CCA TGT TCA AGA ATA ACT ACC C - (5/6-TAMRA).

The following initiator / probe pairs were used for the Taqman analysis (GenBank Accession Number NM_007443 (mouse) for the mouse SF04 sequence):

Direct mouse SF04 primer (Seq ID NO: 7): 5'- GGT ACA ACC TGG CGG TGG -3 '; reverse mouse primer SF04 (Seq ID NO: 8): 5'- GCT CAC GCT CAT CTT GTC CTT AA -3 '; Taqman SF04 mouse probe (Seq ID NO: 9): (5/6-FAM) - TGC CCG TGG CTG AGC CGC - (5/6-TAMRA).

The role of mammalian SF02, SF03, and SF04 in metabolism was further validated by analysis of transcript expression in different tissues.

In Fig. 2, 4, and 6, the relative expression of RNA on the Y axis is shown. In Fig. 2, 4, and 6, the tissues tested are indicated on the X axis. "WAT" refers to adipose tissue White. In Fig. 2, 4, and 6, the panel of wild-type mouse tissues comprises liver, pancreas, muscle, small intestine, WAT, hypothalamus, and heart, and the panel of mouse tissues with control diet comprises liver, muscle, small intestine, wat, brain, and heart.

The function of the proteins SF02, SF03, and SF04 in the metabolism was further validated by analysis of the expression of transcripts in different tissues. Mouse models of insulin resistance and / or diabetes were used, such as mice bearing gene silenced in the leptin pathway (eg, ob / ob mice (leptin)

or db / db mice (leptin / ligand receptor)) to study protein expression. Such mice develop typical symptoms of diabetes, exhibit accumulation of lipids in the liver and frequently have elevated plasma lipid levels (see Bruning J.C. et al. (1998) Mol. Cell. 2: 559-569).

The expression of the mRNAs encoding the described proteins was also examined in susceptible wild-type mice (for example, C57BI / 6) that show symptoms of diabetes, lipid accumulation, and elevated plasma lipid levels, if fed with a high fat diet Studies to determine the expression profile confirm the particular relevance of proteins as regulators of energy metabolism in mammals.

Studies to determine the expression profile confirm the particular relevance of SF02, SF03, and SF04 as regulators of energy metabolism in mammals.

Taqman analysis revealed that SF02 is expressed in several mammalian tissues, exhibiting a maximum level of expression in the liver, and relatively high levels in other tissues, e.g. WAT, small intestine, heart, brain, and muscle. Additionally, SF02 is expressed at lower but still important levels in the hypothalamus and pancreas as depicted in Fig. 2A. The authors of the present invention found, for example, that the expression of SF02 is increasingly regulated in the muscle of ob / ob mice compared to wild-type mice (see Fig. 2B). In wild-type mice fed a high-fat diet, the expression of SF02 is increasingly regulated in the muscle and regulated in a decreasing direction in WAT compared to mice fed with a control diet. The elevated expression of SF02 in active metabolic tissues (eg liver and WAT) and the regulation of gene expression in different mouse models used to study metabolic disorders as described above, suggests that it plays a role in the regulation of Homeostasis of energy.

Taqman analysis revealed that SF03 is expressed in several mammalian tissues, exhibiting the maximum level of expression in WAT and hypothalamus, and relatively high levels in additional tissues, e.g. brain and heart Additionally, SF03 expresses at lower but still important levels in pancreas, muscle, small intestine, and liver as depicted in Fig. 4A. The authors of the present invention found, for example, that the expression of SF03 is regulated in increasing sense in muscle, liver, and small intestine and regulated in decrease direction in the pancreas of ob / ob mice compared to wild-type mice ( see Fig. 4B). In wild-type mice fed a high-fat diet, the expression of SF03 is not regulated. The high expression of SF03 in WAT and in the hypothalamus, which is known to be involved in appetite control, as well as the regulation of gene expression in the mouse model for metabolic syndrome as described above, suggests that it It plays a role in the regulation of energy homeostasis.

The Taqman analysis revealed that SF04 is expressed in several mammalian tissues, showing the maximum level of expression in the liver (Fig. 6A), and lower but still important levels in other tissues, eg, small intestine, heart, muscle, pancreas, WAT, brain, but not in the hypothalamus, as shown in Fig. 6B. The inventors found, for example, that SF04 expression is strongly regulated in increasing sense in the hypothalamus and decreasingly regulated in the heart, muscle, and WAT of ob / ob mice compared to wild-type mice (see Fig. 6C ). In wild-type mice fed a high-fat diet, the expression of SF04 is increasingly regulated in the WAT and decreasingly regulated in muscle, heart, and brain when compared to mice under a control diet. High levels of expression of SF04 in the liver suggest that it plays an essential role in metabolism. The regulation of gene expression in different mouse models used to study metabolic disorders as described above, suggests that it also plays a role in the regulation of energy homeostasis.

Example 4: Analysis of the differential expression of transcripts of the proteins described in human tissues

RNA preparation from human primary adipose tissues was performed as described in the Example.

3. Preparation, hybridization, and scanning of the target were performed as described in the manufacturers manual (see Affymetrix Technical Manual, 2002, obtained from Affymetrix, Santa Clara, USA).

In Fig. 3, 5, and 9, the Y axis represents fluorescence intensity and the X axis represents the time axis. "d0" refers to day 0 (beginning of the experiment), "d12" refers to day 12 of adipocyte differentiation.

Expression analysis (using Affymetrix GeneChips) of genes using differentiation of primary human abdominal adipocytes clearly demonstrates the differential expression of human genes SF02, SF03 and SF13 in adipocytes. Several independent experiments were performed. Experiments show that SF02 and SF03 transcripts are more abundant on day 12 compared to day 0 during differentiation (see Figs. 3 and 5) and that SF13 transcript is very abundant on day 0 compared to day 12 during differentiation. (see Fig. 9). Thus, SF02 and SF03 proteins have to be increased, and SF13 proteins have to be reduced in order for preadipocytes to differentiate into mature adipocytes. The SF02 and SF03 proteins in preadipocytes have the potential to improve fat differentiation, and SF13 protein in preadipocytes has the potential to inhibit fat differentiation. Therefore, the proteins SF02, SF03, and SF13 played an essential role in the regulation of human metabolism, particularly in the regulation of adipogenesis and therefore it could be an essential role in pancreatic diseases (eg diabetes), obesity, and / or metabolic syndrome.

Example 5: In situ hybridizations

Whole and sectional assembly hybridizations were analyzed in situ according to standard protocols that are known to those skilled in the art and as previously described (eg, Pelton, RW et al., (1990) Development 110, 609-620 , Belo, JA et al., (1997) Mech. Dev. 68, 45-57).

The nucleic acid sequence encoding the mouse SF01 protein is expressed in the ventral pancreas (see Fig. 1). Nucleic acid sequences encoding mouse SF05 proteins (see Fig. 7) and mouse SF06 (see Fig. 8) are expressed in the pancreas.

Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be considered unduly limited to such specific embodiments. In fact, various modifications of the modes described for carrying out the invention that are obvious to those skilled in molecular biology or related fields, should be considered to be within the scope of the following claims.

16 LIST OF SEQUENCES

 Protein: NP_114115

 Protein: NP_848759

 Protein: NP_848602

 Protein: NP_057906

 Protein: NP_004475

 Protein: NP_031469

 Protein: NP_001624

 Protein: NP_033276

 Protein: NP_005016

 Protein: NP_766221

 Protein: NP_872317

 Protein: NP_081116

 Protein: NP_006198

 Protein: NP_062670

 Protein: NP_062555

Protein: NP_647456

Protein: NP_644808

 Protein: NP_083844

Protein: NP_002395

Protein: NP_034106 Protein: NP_000090

Protein: NP_034310

 Protein: NP_006477

 Protein: NP_035279

Protein: NP_000933

Claims (9)

one.

Use of a composition comprising an SF06 protein as characterized by SEQ ID Nos: 30 or 32 and / or a nucleic acid molecule encoding an SF06 protein as characterized by SEQ ID Nos: 30 or 32, preferably the molecule being nucleic acid a DNA molecule, more preferably a cDNA or genomic DNA molecule, and optionally pharmaceutically acceptable carriers, diluents, and / or additives for the preparation of an agent for the detection of diseases or dysfunctions selected from the group consisting of diabetes, obesity, and / or metabolic syndrome.

2.

Use of a composition comprising an SF06 protein as characterized by SEQ ID Nos: 30 or 32 and / or a nucleic acid molecule encoding an SF06 protein as characterized by SEQ ID Nos: 30 or 32, said acid molecule being Nucleic preferably a DNA molecule, more preferably a cDNA or genomic DNA molecule, and optionally pharmaceutically acceptable carriers, diluents, and / or additives for the preparation of a medicament for the treatment, relief and / or prevention of diseases or dysfunctions selected from the group consisting of diabetes, obesity, and / or metabolic syndrome.

3.

Use according to claim 1 or 2, wherein the nucleic acid molecule is a mammalian SF06 nucleic acid as characterized by SEQ ID Nos: 30 or 32, which particularly encodes the human SF06 polypeptide as characterized by SEQ ID NO : 32 and / or a nucleic acid molecule, which is complementary to it.

Four.

Use according to any one of claims 1-3, wherein said nucleic acid molecule is selected from the group consisting of

(to)

 a nucleic acid molecule encoding a polypeptide, the polypeptide having a sequence as shown in SEQ ID NO: 30 or SEQ ID NO: 32, or an isoform of the polypeptide according to SEQ ID NO: 30 or SEQ ID NO: 32 ;

(b)

 a nucleic acid molecule comprising or is the nucleic acid molecule according to SEQ ID NO: 30 or SEQ ID NO: 32;

(C)

 a nucleic acid molecule that is degenerated, as a result of the genetic code, with the nucleic acid sequences defined in (a) or (b);

(d)

 a nucleic acid molecule that hybridizes at 50 ° C in a solution containing 1 x SSC and 0.1% SDS to a nucleic acid molecule as defined in claim 3 or as defined in (a) to (c) and / or a nucleic acid molecule that is complementary thereto;

(and)

 a nucleic acid molecule encoding a polypeptide that is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99.6% identical to human SF06 as defined in the claim 3 or to a polypeptide as defined in (a).

5.

Use according to any one of claims 1-4, wherein said nucleic acid molecule is a recombinant nucleic acid molecule and / or a vector, particularly an expression vector and / or a hybridization probe, a primer or a antisense oligonucleotide.

6.

Use according to any one of claims 1-5, wherein the polypeptide is a recombinant polypeptide and / or a fusion polypeptide.

7.

Use according to any one of claims 1-6, for monotherapy or combination therapy together with at least one additional pharmaceutical agent, the at least one additional pharmaceutical agent being selected from the group consisting of 2-amino-1,3- derivatives. propanediol, 2-amino-2- [2- (4-octylphenyl) ethyl] propane-1,3-diol hydrochloride, 40-O (2-hydroxyethyl) -rapamycin, SDZ-RAD, Everolimus, 6- (3- dimethyl-aminopropionyl) -forskolin, 6mercaptopurine (6-MP), A-420983, ABX-CBL (CBL-1), Alefacept, ICAM-1 antisense inhibitor (ISIS 2302), Enlimomab, BIRR1, Alicaforsen, Antithymocyte immunoglobulin (ATG) , Azathioprine, Baohuosido-1, basiliximab, BMS279700, BTI-322, Cladribine, CP-690550, Cyclophosphamide (CTX), Cyclosporine (cyclosporine A, cyclosporine), Daclizumab, HAT (Humanized Anti-Tac), Anti-Tac SMART, Anti-Tac SMART, -CD25, humanized anti-IL2 receptor, Dexamethasone (Decadron, Dexone, Dexasone), DIAPEP-277, boronic acid dipeptide (DPBA), docosahexaenoic acid (DHA), efali zumab, Efomycin M, FTY720 (derivative of oral myriocin), Glatiramer acetate (copolymer-1), Glial Fibrillar acidic protein (GFAP), Gusperimus (15-deoxyspergualin), HLA-B2702 peptide, hu1124 (anti-CD11a), hOKT31y ( Ala-Ala), Infliximab, Interferon, ISAtx247, isotretinoin, L-683,742, Leflunomide (ARAVA), Medi-500 (T10B9), Medi507, Methotrexate, Mitoxantrone, Mycophenolate-Mofetil, OKT4A, Muromonab-CD-4 , Rifampicin, Rituximab, S100�, Sirolimus, Rapamycin, Tacrolimus (Prograf; FK-506), or Triptolide.

8.

Method of identification of a (poly) peptide involved in the regulation of energy homeostasis and / or metabolism in a mammal, comprising the steps of

(to)

contacting a collection of (poly) peptides with an SF06 polypeptide as characterized by SEQ ID Nos: 30 or 32 or an SF06 protein encoded by a nucleic acid selected from the group consisting of:

(i)

a nucleic acid molecule encoding a polypeptide, the polypeptide having a sequence as shown in SEQ ID NO: 30 or SEQ ID NO: 32, or an isoform of the polypeptide according to SEQ ID NO: 30 or SEQ ID NO: 32 ;

(ii)

a nucleic acid molecule comprising or is the nucleic acid molecule according to SEQ ID NO: 30 or SEQ ID NO: 32;

(iii) a nucleic acid molecule that is degenerated, as a result of the genetic code, with the nucleic acid sequences as defined in (i) or (ii);

(iv)

a nucleic acid molecule that hybridizes at 50 ° C in a solution containing 1 x SSC and 0.1% SDS to a mammalian SF06 nucleic acid as characterized by SEQ ID Nos: 30 or 32 or as defined in (i ) to (iii) and / or a nucleic acid molecule that is complementary thereto;

(v)

a nucleic acid molecule encoding a polypeptide that is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99.6% identical to human SF06 as characterized by SEQ ID NO: 32 or to a polypeptide as defined in (i) under conditions that allow the fixation of said or said (poly) peptides;

(b)

remove the (poly) peptides that are not fixed and

(C)

identifying the (poly) peptides that bind to said SF06 polypeptide as characterized by SEQ ID Nos: 30 or 32 or to said SF06 protein encoded by a nucleic acid selected from the group consisting of

(i)

a nucleic acid molecule encoding a polypeptide, the polypeptide having a sequence as shown in SEQ ID NO: 30 or SEQ ID NO: 32, or an isoform of the polypeptide according to SEQ ID NO: 30 or SEQ ID NO: 32 ;

(ii)

a nucleic acid molecule comprising or is the nucleic acid molecule according to SEQ ID NO: 30 or SEQ ID NO: 32;

(iii) a nucleic acid molecule that is degenerated, as a result of the genetic code, with the nucleic acid sequences defined in (i) or (ii);

(iv)

a nucleic acid molecule that hybridizes at 50 ° C in a solution containing 1 x SSC and 0.1% SDS to a mammalian SF06 nucleic acid as characterized by SEQ ID NOs: 30 or 32 or as defined in (i) a (iii) and / or a nucleic acid molecule that is complementary thereto;

(v)

a nucleic acid molecule encoding a polypeptide that is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99.6% identical to human SF06 as characterized by SEQ ID NO: 32 or to a polypeptide as defined in (i) under conditions that allow the fixation of said or said (poly) peptides.

9.

Method for screening an agent, which affects / modulates the activity of an SF06 polypeptide as characterized by SEQ ID NOs: 30 or 32, comprising the steps of

(a) incubating a mixture comprising (aa) an SF06 polypeptide as characterized by SEQ ID NO: 30 or 32; Y (ab) a candidate agent under conditions in which said SF06 polypeptide or fragment thereof exhibits a reference activity,

(b)

detecting the activity of said SF06 polypeptide to determine an activity for the agent; Y

(C)

determine a difference between the activity for the agent and the reference activity.

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